Kinase signaling pathways are linked to most cellular functions, so understanding their activation is crucial to finding new drugs and using therapeutic inhibitors to treat patients. Current kinase activity measurements are not suitable to analyze large numbers of cells on an individual basis. Here, a microfluidic device will be developed to enable direct determination of protein kinase activation in single cells. The device will be applied to the analysis of mutant kinase BCR-ABL activity in chronic myelogenous leukemia (CML) cells. Three aims are proposed: 1.Demonstrate separation and detection of fluorescently labeled peptide substrates for BCR-ABL kinase on a PDMS microchip device. 2.) Learn to culture cells, load them with fluorescent dyes and peptide substrates, and lyse them with a laser in the device. 3.) Combine steps 1 and 2 by attempting to separate and detect the contents of cells that have been loaded with peptide substrates and lysed with a laser in the microchip. In Aim 1, microchip fabrication and surface treatment will be performed to make a functional device for electrophoresis that is resistant to cell adhesion. Separation and detection of peptide substrates will be optimized. In Aim 2, the candidate will master culture and care of cells and loading with fluorescent dyes and peptide substrates. Cells will be tested to determine if the microchip environment affects viability. A laser-based cell lysis method will be optimized on-chip. In Aim 3, Aims 1 and 2 will be combined and key parameters tested/optimized for best performance. BCR-ABL expressing and control cells will be analyzed with the device in a series of experiments designed to explore its capability. The proposed work will use new microchip technology to achieve rapid and inexpensive monitoring of chronic myelogenous leukemia (CML) patients being treated by kinase inhibitor drugs. The clinical device would provide oncologists with better information to make individually tailored "drug cocktail" treatments possible, and would aid medical research efforts towards understanding and treatment of CML. The device could also be used for research and therapy for many other diseases caused by cell signaling problems. [unreadable] [unreadable] [unreadable]